Cytochrome P450 (CYP) is one of the main metabolic systems involved in xenobiotic clearance in live organisms. Its activity is mainly carried out at hepatic level, but in the last two decades, its function has been also reported to be important in other extrahepatic tissues, especially in the central nervous system. In brain, CYP isoenzymes vary their expression depending on the brain area and the cell type. However, the role of the different isoforms is not yet well characterized. Some of them have been involved in detoxification and/or toxic activation of xenobiotics; while other have been related with production of oxidative stress through the metabolism of endogenous compounds. Therefore, the presence and the function of the CYP in the brain has been related with neurodegenerative diseases such as Parkinson’s Disease (PD). On the other hand, the study of the degeneration of dopaminergic neurons in vitro has been carried out by the use of several toxins that promote apoptosis and similar pathological features that the observed in PD. Among these xenobiotics, 1-methyl-4-phenylpyridinium (MPP+), rotenone, and paraquat are the most used because they are able to promote neurodegeneration of dopaminergic cells by directly targeting complex I of mitochondria. Recently, it has been reported that some CYP isoforms, such as CYP 2D6, can be involved in the development of this neurodegenerative disease. To better characterize this role, we have studied the induction of some isoforms in an in vitro system. Undifferentiated SH-SY5Y cells were treated with well-known inducers of CYP for 48 hours, namely β-naphtoflavone (β-NF), ethanol (EtOH), and cyclophosphamide (CPA); and qRT-PCR, Western Blot (WB) and confocal microscopy analysis were performed. CPA increased the mRNA levels of CYP 1A1, 2D6 and 2E1, while the other inducers promoted a slight increase on these isoforms compared to CPA. WB analysis confirmed the induction promoted by CPA in CYP 1A1 and 2D6, and revealed that CYP 2D6 was also inducible by EtOH. Moreover, CYP 2E1 was increased by β-NF and EtOH treatments. In differentiated SH-SY5Y cells, a preliminary WB analysis of CYP 2D6 and 2E1 was also performed. The results suggested a change in the regulation of the expression of CYP 2D6 when treated with β-NF, and also of CYP 2E1 levels when treated with β-NF and CPA. Immunohistochemistry analysis confirmed the inducibility of CYPs and showed a co-localization of CYP 2D6 with mitochondria. These data indicate that CYP can be induced in both undifferentiated and differentiated neuroblastoma cells, and underline the possibility to use this in vitro system for studying the role of CYPs in neurodegeneration. Moreover, as showed by our group and others, β-naphtoflavone and ethanol have the ability to induce the expression of some CYP isoforms. In order to study the possible role of CYP induction in neurodegeneration, we have use the same in vitro model than before, where undifferentiated neuroblastoma SH-SY5Y cells have been treated with these inducers separately previous and during exposure of MPP+. In both experimental conditions, the toxic effect of MPP+ was partially reverted. MTT assays showed an increase in cell viability of approximately 17% in β-NF treatment, while EtOH showed an increase between 13-15%. The analysis of apoptotic population by flow cytometry showed that both treatments were able to restore the populations to control values compared to the toxic effect of MPP+. The mitochondrial fission-fusion kinetics revealed that both treatments were able to avoid the impairment of this mitochondrial motility after exposure to the toxin. Finally, this neuroprotection was confirmed by a lowest effect of MPP+ upon complex I activity when cells were preincubated with the inducers. These results bring new insights about the possible role of CYPs, specially CYP 2D6, in neuroprotection and possible development of therapeutic drugs.

FERNANDEZ ABASCAL, J. (2018). Role of cytochrome P450 against toxic insult in neuroblastoma SH-SY5Y cells.

Role of cytochrome P450 against toxic insult in neuroblastoma SH-SY5Y cells

FERNANDEZ ABASCAL, JESUS
2018-01-01

Abstract

Cytochrome P450 (CYP) is one of the main metabolic systems involved in xenobiotic clearance in live organisms. Its activity is mainly carried out at hepatic level, but in the last two decades, its function has been also reported to be important in other extrahepatic tissues, especially in the central nervous system. In brain, CYP isoenzymes vary their expression depending on the brain area and the cell type. However, the role of the different isoforms is not yet well characterized. Some of them have been involved in detoxification and/or toxic activation of xenobiotics; while other have been related with production of oxidative stress through the metabolism of endogenous compounds. Therefore, the presence and the function of the CYP in the brain has been related with neurodegenerative diseases such as Parkinson’s Disease (PD). On the other hand, the study of the degeneration of dopaminergic neurons in vitro has been carried out by the use of several toxins that promote apoptosis and similar pathological features that the observed in PD. Among these xenobiotics, 1-methyl-4-phenylpyridinium (MPP+), rotenone, and paraquat are the most used because they are able to promote neurodegeneration of dopaminergic cells by directly targeting complex I of mitochondria. Recently, it has been reported that some CYP isoforms, such as CYP 2D6, can be involved in the development of this neurodegenerative disease. To better characterize this role, we have studied the induction of some isoforms in an in vitro system. Undifferentiated SH-SY5Y cells were treated with well-known inducers of CYP for 48 hours, namely β-naphtoflavone (β-NF), ethanol (EtOH), and cyclophosphamide (CPA); and qRT-PCR, Western Blot (WB) and confocal microscopy analysis were performed. CPA increased the mRNA levels of CYP 1A1, 2D6 and 2E1, while the other inducers promoted a slight increase on these isoforms compared to CPA. WB analysis confirmed the induction promoted by CPA in CYP 1A1 and 2D6, and revealed that CYP 2D6 was also inducible by EtOH. Moreover, CYP 2E1 was increased by β-NF and EtOH treatments. In differentiated SH-SY5Y cells, a preliminary WB analysis of CYP 2D6 and 2E1 was also performed. The results suggested a change in the regulation of the expression of CYP 2D6 when treated with β-NF, and also of CYP 2E1 levels when treated with β-NF and CPA. Immunohistochemistry analysis confirmed the inducibility of CYPs and showed a co-localization of CYP 2D6 with mitochondria. These data indicate that CYP can be induced in both undifferentiated and differentiated neuroblastoma cells, and underline the possibility to use this in vitro system for studying the role of CYPs in neurodegeneration. Moreover, as showed by our group and others, β-naphtoflavone and ethanol have the ability to induce the expression of some CYP isoforms. In order to study the possible role of CYP induction in neurodegeneration, we have use the same in vitro model than before, where undifferentiated neuroblastoma SH-SY5Y cells have been treated with these inducers separately previous and during exposure of MPP+. In both experimental conditions, the toxic effect of MPP+ was partially reverted. MTT assays showed an increase in cell viability of approximately 17% in β-NF treatment, while EtOH showed an increase between 13-15%. The analysis of apoptotic population by flow cytometry showed that both treatments were able to restore the populations to control values compared to the toxic effect of MPP+. The mitochondrial fission-fusion kinetics revealed that both treatments were able to avoid the impairment of this mitochondrial motility after exposure to the toxin. Finally, this neuroprotection was confirmed by a lowest effect of MPP+ upon complex I activity when cells were preincubated with the inducers. These results bring new insights about the possible role of CYPs, specially CYP 2D6, in neuroprotection and possible development of therapeutic drugs.
2018
FERNANDEZ ABASCAL, J. (2018). Role of cytochrome P450 against toxic insult in neuroblastoma SH-SY5Y cells.
FERNANDEZ ABASCAL, Jesus
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/1051261
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